System and methods for ionizing compounds using matrix-assistance for mass spectrometry and ion mobility spectrometry

ABSTRACT

An ionization method for use with mass spectrometry or ion mobility spectrometry is a small molecule compound(s) as a matrix into which is incorporated analyte. The matrix has attributes of sublimation or evaporation when placed in vacuum at or near room temperature and produces both positive and negative charges. Placing the sample into a region of sub-atmospheric pressure, the region being in fluid communication with the vacuum of the mass spectrometer or ion mobility spectrometer, produces gas-phase ions of the analyte for mass-to-charge or drift-time analysis without use of a laser, high voltage, particle bombardment, or a heated ion transfer region. This matrix and vacuum assisted ionization process can operate from atmosphere or vacuum and produces ions from large (e.g. proteins) and small molecules (e.g. drugs) with charge states similar to those observed in electrospray ionization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Provisional Application Ser. No.61/649,393 filed May 21, 2012, Provisional Ser. No. 61/684,606 filedAug. 17, 2012, and Provisional Ser. No. 61/757,040 filed Jan. 25, 2013.

FIELD OF DISCLOSURE

The disclosed systems and methods relate to mass spectrometry (MS) andion mobility spectrometry (IMS). More particularly, the disclosedsystems and methods relate to ionization facilitated by a small moleculematrix and sub-atmospheric pressure conditions.

BACKGROUND OF THE DISCLOSURE

Compounds are currently ionized in vacuum (herein defined as lowpressures relative to atmospheric pressure) ion sources of a massspectrometer by first evaporating the analyte followed by gas-phaseionization as in electron ionization or chemical ionization, or by laserablation of the analyte either directly or in a small molecule(chemical) matrix as in matrix-assisted laser desorption/ionization(MALDI), or by use of a high velocity particle or ion as in secondaryionization mass spectrometry (SIMS) and fast atom bombardment (FAB), orby methods such as field desorption where a high voltage is placed onemitters having sharp edges or tips to generate ions, or thermosprayionization where a solution flowing into a low pressure region is heatedto rapidly effect vaporization and ionization or by placing a voltage ona solution flowing from a capillary as in electrospray ionization (ESI).All of these methods require a high energy means of producing gas-phaseions for mass analysis of analyte.

The current practice for analysis of compounds which cannot be vaporizedwithout destruction by heat is to use MALDI or ESI, and variantsthereof, or the newly developed methods of inlet ionization termedlaserspray ionization inlet (LSII), solvent assisted ionization inlet(SAII), and matrix assisted ionization inlet (MAII), and vacuumionization methods termed laserspray ionization vacuum (LSIV).

MALDI is typically used with a time-of-flight (TOF) mass spectrometerand is commonly referred to as MALDI-TOF. The source region operates atvery low pressure (high vacuum). MALDI uses a small molecule matrix suchas 2,5-dihydroxybenzoic acid (2,5-DHB) to facilitate ionization ofnonvolatile analyte but requires an expensive laser and extractionvoltage, and produces mostly singly charged ions. MALDI-TOF instrumentsare costly and dedicated to MALDI analysis. Limitations of MALDI includehigh matrix related background, hot/cold spot issues leading toirreproducibility and thus are not readily applicable to extractingquantitative data. Intermediate pressure MALDI sources, operating in themilli-Torr and sub-milli-Torr pressure range, can be interfaced withinstruments that are multipurpose and provide high sensitivity analysis,but these ion sources and associated lasers are also expensive. Avariant of MALDI called atmospheric pressure MALDI produces ions atatmospheric pressure before entering the mass spectrometer inlet, whichin the presence of the extraction voltage cause loss of ions at theinlet aperture (rim loss). Atmospheric pressure MALDI sources areavailable that operate on instruments designed for electrosprayionization (ESI) but are less sensitive than vacuum MALDI. Because MALDIproduces primarily singly charged ions, the intermediate pressure andatmospheric pressure MALDI sources interfaced with instruments havinglimited mass-to-charge (m/z) range for singly charged ions limits theutility of the method to compounds within the limited mass range of theinstrument. Therefore, for analysis of intact, high-mass compounds suchas proteins, the MALDI-TOF instrument, with unlimited mass range isrequired. Contrary, proteins and protein complexes can be digested byenzymes and analyzed using high performance mass spectrometers (as ine.g., ‘bottom up’, ‘shot gun proteomics’) at the expense of the intactanalyte information, time, cost, and expertise. Small molecule analysisis limited by the matrix background in competition with the ionizationof the desired analyte (e.g., drugs and metabolites) at any of thepressure regimes used in MALDI. MALDI requires a laser and for vacuumMALDI, sample introduction from AP to vacuum is time consuming andrequires expensive instrument modifications. Typical lasers for use inMALDI use laser fluencies of generally between 2 and 60 kJ m⁻². Inducingfragmentation using collision-induced dissociation (CID) of singlycharged ions produces little sequence (e.g., peptides) information,especially of fragile posttranslational or other chemical modificationsthat frequently are lost preventing structural information of theanalyte to be obtained. Newer and improved fragmentation methods such aselectron transfer dissociation (ETD) and electron capture dissociation(ECD) are not applicable to singly charged analyte ions. Because of theuse of a laser, MALDI is a harsher method relative to, for example, LSI(laser fluencies of generally between 40 and 150 kJ m⁻² with the laseraligned in transmission geometry) or ESI limiting applications to moresturdy, less fragile molecules and rarely to the analysis of e.g.,molecular non-covalent complexes, MALDI can analyze molecular complexesafter chemical crosslinking the complex prior to MALDI mass analyses.

One-Dimensional (1-D) and 2-D gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and agarose gelsare used in protein and deoxyribonucleic acid (DNA)/ribonucleic acid(RNA) separation and purification, and which can be coupled with ESI andMALDI-MS as well as ion mobility-MS and MS/MS but indirectly bydigesting the macromolecule(s) in the gel slice and subsequentlysolution extracting it from the gel environment. Nevertheless, MSovercomes the need for specific and expensive antibodies for detectionusing, for example, Western blots; many compounds do not have specificantibodies and therefore cannot be detected. More commonly applied areliquid chromatography (LC)-MS and MS/MS or ion mobility-MS approachesfor additional separation. Common to both ESI and MALDI, and the methodsderived from them, are ion suppression issues, problems with thepresence of salt, and robustness.

MALDI operates from the solid state and is a surface method enablingmolecular surface imaging approaches to determine the localization ofcertain analytes within a surface. A voltage is applied, frequentlyseveral kilovolts, to lift the ions from the surface and accelerate themto the analyzer. The matrix requires having sufficient absorption at thelaser wavelength used to enable matrix desorption from the surface.Analyte ionization occurs in a region very near the matrix surface (<100microns from the surface). A notable degree of chemical backgroundassociated with the desorption process creates significant backgroundnoise especially in the low mass region (<800 m/z). MALDI is thereforelimited in this mass range for which applications range from drugdevelopment (clinical applications) to forensic analyses. To increasethe speed, especially for imaging applications, expensive highrepetition lasers can be employed to desorb/ionize more rapidly enablethe measurement of summed mass spectra from ˜100 laser shots and, incase of imaging of surfaces, ions in each mass spectrum are used todetermine analyte location employing respective computing programs.Thus, the mass spectra generated from ˜100 laser shots are summed into asingle mass spectrum which represents one pixel in the image. Advantagesof the MALDI method is that predominantly singly charged ions areproduced so that interpretation is simplified, which is important forcomplex mixtures.

ESI is an ionization method whereby a voltage, usually several thousandvolts, is placed on a capillary through which a solution is passedrelative to a counter electrode which contains the inlet entrance to thevacuum of the mass spectrometer. Highly charged liquid droplets areformed in the ESI process and desolvation of these droplets leads toformation of bare ions that are analyzed by the mass spectrometer. Whilethe MALDI method produces primarily singly charged ions, the ESI liquidintroduction method produces ions of high charge states when multipleionization sites exist on the analyte molecule. ESI methodology is notwell suited for surface analysis, although a method called desorptionelectrospray ionization (DESI) can sample surfaces but with rather poorspatial resolution and limited in upper molecular weight range ofnonvolatile compounds. A newer variation, nano-DESI, enables improvedspatial resolution measurements at the expense of critical alignment andexpertise. Combined methods of laser ablation of a surface with captureof the ablated material in the ESI plume can also be used to imagesurfaces.

Because ESI produces multiply charged ions, the method is useful withhigh performance mass spectrometers having limited mass range and themultiply charged ions provide improved fragmentation efficiency usinge.g., CID, ECD, and ETD relative to singly charged ions making analyseson high performance mass spectrometers with limited m/z range suitable.To increase analyte charge, small amounts (frequently <10% volume) ofso-called supercharging reagent can be added to the solution instead oradditionally to other reagents such as acids. Detection of multiplycharged ions relative to singly charged ions is more efficient, as isthe ion mobility gas-phase separation of analyte ions. However, ESIrequires the analyte to be in a suitable solvent to provide “sprayable”conditions. ESI is softer than MALDI making it applicable to analyzingprotein complexes with suitable solvent conditions applied. ESI can becombined with “online” LC for pre-separation applicable for solubleanalyte samples. This approach is not applicable for solubilityrestricted or insoluble analytes or where spatial and temporalresolution matters.

Numerous ionization approaches under the terminology ambient ionizationhave been developed to circumvent some of the problems associated withESI and MALDI. All ambient ionization methods capable of ionizingnonvolatile compounds are variants of ESI and MALDI and while they offeradvantages for certain analyses, they all increase the complexity of theion source. None of these methods offers a simple means of rapidlyintroducing analyte for conversion to gas-phase ions for analysis by MS.All these methods require use of high voltage, lasers, or other sourcesrequiring application of energy or force to the sample. Further, thecurrent means are not well suited for automated high throughput analysisbecause of expense or problems associated with robustness of themethods.

New ionization methods have recently been introduced. Inlet ionizationmethods used in MS include laserspray ionization inlet (LSII), matrixassisted ionization inlet (MAII), and solvent assisted ionization inlet(SAII). All of these methods produce abundant highly charged ionswithout the use of a voltage from the solid state (MAII, LSII) orsolution (SAII). Ionization occurs in a heated channel (inlet) thatconnects a higher pressure region (typically atmospheric pressure) and alower pressure region (typically the first vacuum region of a massspectrometer. In practice, the matrices or solvents disclosed for thesemethods require that the channel be heated to greater than 150° C. andanalyte ion abundance reaches a maximum between 250 and 450° C. Ionabundances reported for these methods are not analytically useful below150° C. The mechanism of ionization of the inlet ionization methods ispurported to involve creation of droplets of matrix or solvent withinthe heated channel with an excess of one charge at the droplet surfaceand an excess of the opposite charge in the bulk of the droplet. Removalof the surface layer by for example superheating the droplets as theytraverse from the high to the low pressure regions with rapid bubblingon nucleation of the droplet. However the analyte ions are formed, theionization event occurs inside the heated channel linking a higher and alower pressure region.

With LSII, a laser ablates the matrix with incorporated analyte, thesample, into the heated inlet where ionization occurs. In MAII, thesample is introduced physically into the heated channel producingidentical ionization as LSII with the same sample. In SAII, a solventreplaces the small molecule matrix and similarly produces analyte ionswhen introduced into the heated channel. In all inlet ionizationmethods, similar to MALDI, the matrix, or solvent, is present in thesample in orders of magnitude higher molar ratio relative to theanalyte.

LSII-MS is a surface method that has the potential to characterizemacromolecular structures directly from their native and complexenvironment with high spatial resolution important in surface imaging,similar to MALDI, but producing abundant highly charged ions as long asheat can be applied to the inlet tube. Mass spectrometers with skimmercone inlets have been retrofitted with a heatable inlet tube to produceanalytically useful analyte ions for analysis by mass spectrometry.Contrary to MALDI, LSII does not require the absorption by the matrixcompound at the laser wavelength. The laser can create a shockwave sothat the matrix:analyte association is ablated into the heated inlettube. LSII was introduced on high performance mass spectrometersoperating at atmospheric pressure without the application of anyelectrical field demonstrating its usefulness for tissue analysis andsurface imaging.

A requirement for all of the inlet ionization methods is a heated inlettube linking atmospheric pressure and the vacuum of the massspectrometer, requiring the inlet channel be heated to greater than 150°C. for analytically useful results, especially when organic matrices areused as in MAII or LSII. Most mass or ion mobility spectrometer ionsources are not equipped with such a heated inlet tube and must beretrofitted. A large number of small molecule compounds have been shownto produce multiply charged mass spectra of peptides and small proteinsat inlet temperature >400° C. Even so, the matrices reported for MAIIand LSII that require a hot inlet to produce ions in good yield are alsosufficiently nonvolatile that they collect on ion transmission elementswithin the instrument causing contamination over time and loss ofinstrument sensitivity. More volatile matrix compounds would alleviatethis problem and a few (1,4-dihydroxy-2,6-dimethoxybenzene (DHDMB),salicylamide, 3,4-dihydroxyacetophenone, mono-methylfumarate,4-trifluoromethylphenol) were discovered that produce analyticallyuseful gas phase analyte ions when introduced into a heated inletchannel when the channel was heated to less than 150° C. and as low as50° C. Some of these matrices (e.g., DHDMB) produced multiply chargedanalyte ions when introduced to a skimmer cone inlet using a sourceblock temperature of 150° C., or by laser ablation of the sample invacuum. This latter vacuum method in which an inlet channel is notavailable and the only energy or force supplied to the sample is fromthe laser is called laserspray ionization vacuum (LSIV).

LSIV was introduced on vacuum sources offering advantages anddisadvantages described above for MALDI (vacuum source) except that ithas an additional advantage of producing multiply charged ions fromanalyte similar to SAII, LSII, MAII, and ESI. Conditions for LSIVoperation combine the requirements of LSII and vacuum MALDI requiringsufficient absorption of the matrix at the wavelength of the laser usedand in a timeframe that allows the matrix to be removed from thematrix:analyte association by desolvation which are stringentexperimental conditions. Similar to MALDI, fragile gangliosides cannotbe analyzed using LSIV, contrary to LSII. The most prominent LSIV matrixat intermediate pressure is 2,5-dihydroxyacetophenone (2,5-DHAP), alsoused as a MALDI and LSII matrix. The most prominent LSIV matrix at lowpressure (high vacuum) is 2-nitrophloroglucinol(2-nitro-benzene-1,3,5-triol, 2-NPG). In LSIV, a laser is used todesorb/ablate the sample from vacuum conditions to form multiply chargedions directly from surfaces in intermediate pressure and low pressuremass spectrometer ion sources. Without a means of supplying heat todesolvate the charged matrix:analyte particles or droplets produced bylaser ablation, LSIV is limited in its ability to analyze highermolecular weight proteins. The largest protein detected to date on anintermediate pressure source is lysozyme, molecular weight 14300, withcharge states identical to ESI and inlet ionization methods using thesame Waters SYNAPT G2 (Quadrupole ion mobility spectrometry (IMS) TOFmass spectrometer with a 8000 m/z upper limit). With a low pressureMALDI-TOF source, LSIV produces multiply charged ions of carbonicanhydrase.

The method described herein differs from and improves the abovedescribed ionization methods. This method produces multiply charged ionsby using a matrix that when exposed to sub-atmospheric pressureconditions spontaneously produces analyte ions of charge states similarto the inlet ionization methods, but without the need of a heatedchannel or a force that allows the matrix:analyte association to enterthe gas phase. Unlike with inlet ionization, the initial ionizationevent occurs from the surface of the substrate upon which thematrix:analyte, the sample, is placed by exposing the sample tosub-atmospheric pressure available with any mass spectrometer. The MAIVprocess is spontaneous and continues without the application of a force.No external energy is necessary, so that ionization is initiated by theenergy already in the system. This initial ionization event is notdependent on a heated channel, but is affected by heat, which can beabove or below room temperature, applied to the substrate, and requiresexposure to sub-atmospheric pressure to initiate the ionization event.Thus, simply placing the sample using an appropriate matrix compoundinto a vacuum ion source of a mass spectrometer initiates the ionizationevent that produces abundant analyte ions. Likewise, by placing thesample in sub-atmospheric pressure conditions using an atmosphericpressure ion source inlet, such as is used with ESI, using anappropriate matrix the ionization event is initiated spontaneously. Ineither case, heating or cooling the substrate onto which the sample isplaced, using methods known to those practiced in the art, extends thecompounds that spontaneously produce ions from the method describedherein. This method will be referred to as matrix assisted ionizationvacuum (MAIV) and matrices that produce analyte ions by this method foranalysis by mass spectrometry or ion mobility are referred to as MAIVmatrices. MAIV is usefulness for tissue analysis and surface imaging ofsuch as those of endogenous and exogenous origin and examples includedrugs, metabolites, pesticides, lipids, peptides, proteins, chemicallyor posttranslational modified peptides or proteins, protein complex,receptors, ligands, catalysts, carbohydrates, glycans, antibodies,biomarkers, and other compounds produced by synthesis, such as syntheticpolymers, on mass range limited mass spectrometers. These analytes canbe pure or present in biological/synthetic environments such as urine,blood, skin, tissue sections, biofilms, eatable goods, flesh, vegetablesurfaces, drug pills, bacterial, microbial, artificial bone,archaeological artifacts, painting, or synthetic polymer films, andothers. The production of highly charged ions directly from surfaces ina soft manner and in high abundance allows sequencing of for examplepeptides and proteins using for example ETD.

SUMMARY OF THE DISCLOSURE

Embodiments disclosed herein provide systems and methods for producingions, singly or multiply charged, and analyzing compounds of widelyvarying molecular weights including small molecules as well asmacromolecules using matrices and vacuum and referred to as matrixassisted ionization vacuum (MAIV). We disclose here the ability toincorporate analyte into a matrix, or into a solvent in which the MAIVmatrix is dissolved, called the sample, to produce ions from the analytewhen the sample is introduced into a sub-atmospheric pressure (vacuum)region of a mass spectrometer or ion mobility spectrometer. With thismethod, analyte ions are produced without need of any high energy sourcesuch as a laser, high voltage, or high velocity particle for iongeneration. The ions produced by this method have charge states similarto ESI, LSII, LSIV, and MAII. Thus, proteins are multiply charged andfall within the m/z range of mass spectrometers commonly used in MS.

In MAIV, analyte ions are created at the surface of a sample holder orsubstrate without the use of a laser or voltage producing ions in chargestates similar to ESI. The MAIV the sample can be manipulated fromatmospheric pressure or from the vacuum of common ion sources but doesnot necessitate an ion source now common with all mass spectrometers.The ion source can be eliminated using only an inlet to the massspectrometer which can be simply a pin-hole leak. The diameter size ofthe hole and the type of substrate define the applications. In any caseionization is through exposure to sub-ambient conditions in which thesample is in fluid contact with sub-ambient pressure of the mass or ionmobility analyzer. When the sample is exposed to sub-atmosphericpressure, ionization commences spontaneously and is continuous until thematrix in the sample is depleted or, for multiple sample, the samplesubstrate is moved to the next the sample or removed from thesub-atmospheric pressure region. Ionization can be prolonged by, forexample, cooling the substrate, or intensified but for a shorterduration by heating the substrate. From the same sample through changesof the voltages, especially in the source region, multiply and singlycharged MAIV ions can be formed of peptides at will.

The MAIV matrices so far discovered when placed in sub-atmosphericpressure, or heated to less than 150° C., or placed in vacuum and heatedto less than 100° C., splinter matrix particles from the surface. Theseparticles have charge. It is therefore postulated that this splinteringprocess initiates the ionization event, but ionization directly from thematrix surface is possible. The charged particles splintered from thesurface may undergo further splintering producing smaller chargedparticles. The splintered particles have either excess positive orexcess negative charge.

Similar to all ionization methods that produce analyte ions from chargeddroplets or particles, a process is necessary to remove the matrix orsolvent, commonly referred to as desolvation. In ESI a heated gas orheated inlet tube are used to desolvate the droplet and release the bareanalyte ion. With some MAIV matrices, no external energy is required toproduce the bare analyte ions. However, by providing external energy, awider range of compounds act a MAIV matrices spontaneously producingcharge particles which, when desolvated, produce the bare analyte ions.Means of enhancing evaporation of the sample as well as subsequentdesolvation of charged particles from MAIV matrices include radiative(e.g. infrared (IR), visible, ultraviolet (UV)) or convective heat,microwave and radiofrequency radiation, collisions with gas or surface,and other means known to those practiced in the art. Frequently,improved desolvation increases the ion abundance observed and enhancesCID and ETD fragmentation. Therefore, MAIV matrices evaporate or sublimeunder sub-atmospheric pressure at temperatures below 150° C. and ideallybelow 75° C.

According to the above postulates the MAIV matrix must splinter offparticles from the matrix surface that have excess positive or negativecharge. One means of producing a charge when a surface cracks(splinters) is through the mechanism that produces triboluminescence orlight when a crystal is crushed. The light, or lightening is caused by adischarge between cracked surfaces that have opposite charges. Some ofthe known MAIV matrices are also known to triboluminescence. The abovemechanistic discussion is meant to provide insight and if not found tobe correct in its entirety in the future, will not alter the claims ofthis application.

With a MAIV matrix, the sample can be exposed or inserted tosub-atmospheric pressure of a mass spectrometer or ion mobilityspectrometer, by default available, and analyte ions (of positive andnegative charge, hereafter referred to positive and negative ions) areproduced without a laser or a high voltage. Voltages are used to guidethe ions to and through the analyzer and to the detector.

MAIV matrices produce ions spontaneously when exposed to sub-atmosphericpressure. Some MAIV matrices spontaneously produce analyte ions atambient temperature and others require the matrix be heated, typicallyby heating the substrate to less than 150° C. and preferably less than100° C. at or near atmospheric pressure. With MAIV matrices thatevaporate or sublime at sub-atmospheric pressure only when heat isapplied to the matrix or its substrate, desolvation energy usuallyenhances ion abundance. The MAIV ion formation is continuous as in ESI.These matrices may benefit from added heat/cold to a channel connectingtwo different pressure regions, or to the sample substrate (the sampleholder), but unlike reports for inlet ionization (especially MAII), inwhich optimization was reported to occur between about 250 and 450° C.,MAIV matrices optimize at temperatures between −80° C. and +150° C. (ifno sign is provided, the temperature is above 0° C.), and produceanalytically useful analyte ionization between room temperature andabout 75° C. at or near atmospheric pressure. The optimalpressure/temperature values change similarly to the pressure/temperaturephase diagram associated with a matrix compound. Also, unlike inletionization an inlet (e.g., a channel or inlet tube, or skimmer is not arequirement and the ionization event is not initiated by using a forceto transfer the matrix:analyte sample into the inlet.

The substrate with the MAIV the sample can be mechanically introduced toa vacuum source similar to solid probe introduction in electron andchemical ionization methods or sample plate introduction in analyteionization commences as soon as the sample experiences thesub-atmospheric conditions without the use of a laser or the use of aheated channel. Spontaneous ionization can be controlled throughtemperature control of the substrate surface, the use of matrixcombinations, or limiting the exposure to the vacuum conditions by, forexample, placing the sample within a capillary that allows only a smallsurface area of the sample to experience the sub-atmospheric pressure.Because some of the MAIV matrices optimize at or around ambienttemperature and use no high voltage or laser, in principle, any massspectrometer with any ion source or inlet configuration is operationalwith MAIV. Commercially available inlets of atmospheric pressure ionsources (e.g., skimmer or inlet tube), with or without modifications,can be exposed to the operator without need of the ion source enclosurebecause the inlet for these MAIV matrices can be near ambienttemperature and without application of more than a few volts forfocusing the ion beam. Further, the only requirement for ionizationbeing the vacuum inherent in the operation of the analyzer, the energynecessary to operate the instrument for the sample ionization isreduced. Thus, this safe and low energy requirement ionization method isideal for field portable instruments as well as for fragile compounds(protein complexes, posttranslational protein modifications such asphosphorylations, fragile chemical modifications such as catalysts, organgliosides containing fragile sialic acid modifications are examplesthat tend to fragment or explode using high energy ionization sources(MALDI). Further, the simplicity and robustness of the MAIV method makesit potentially useful for clinical analyses.

The disclosed system and method comprises placing a matrix withincorporated analyte in fluid contact with a region of sub-atmosphericpressure, typically generated by the pumping system of an ion mobilityor mass spectrometer. This MAIV method is applicable to vacuum ionsources such as used with matrix-assisted laser desorption/ionization(MALDI) or with electron (EI) or chemical ionization (CI), andatmospheric pressure ionization (API) sources used with electrosprayionization (ESI) or atmospheric pressure chemical ionization (APCI), butdoes not require a laser as in MALDI, added heat as in EI, CI, or APCIto vaporize volatile compounds, or a high voltage as used in ESI and inMALDI. Methods are described that are capable of producing abundantsingly or multiply charged ions for use in MS and ion mobility-MS andMS/MS using fragmentation methods including collision-induceddissociation (CID), electron transfer dissociation (ETD), and electroncapture dissociation (ECD) as examples. The systems and methods can bemodified for certain advantages by using smaller or multiplexedsurfaces/containers, and high throughput analyses using matrices withlow thermal requirements for analyte ion formation, or with the additionof heat or cooling supplied to the sample surface or substrate, or tothe inlet region, or by additional of a gas slow (preferably air) inclose proximity to the sample or aimed at the sample to provide enhancedion abundance to obtain structural characterization using (CID) and highperformance fragmentation such as ECD or ETD known to those practiced inthe art.

This method allows analysis of compounds having a wide range ofvolatility as well as compounds of widely differing molecular weights bysimply introducing the analyte in an appropriate matrix into a vacuumregion of a mass spectrometer. For example, bovine serum albumin (BSA),molecular weight 66 kDa, produces abundant multiply charge molecularions using the matrix 3-nitrobenzonitrile (3-NBN) with the sample(matrix:analyte) placed in solution on e.g., a traditional MALDI plate,dried (although the drying step is not necessary as it can occur withinthe vacuum system), or, alternatively the matrix and analyte can beground together (the sample prepared ‘solvent-free’; it should beunderstood that the phrase “solvent-fee” covers means that avoids theuse of a solvent during matrix deposition), and inserted into theintermediate pressure MALDI source of, for example, a Waters CorporationSYNAPT G2 mass spectrometer. Highly charged protonated molecular ionsare observed and fragment ions can be generated, especially from highlycharged ions using CID, ETD, ECD and variations thereof. Other MAIVmatrices include 2-nitrobenzonitrile (2-NBN),5-methyl-2-nitrobenzonitrile, coumarin, methyl-2-methyl-3-nitrobenzoate,methyl-5-nitro-2-furoate, bronopol (2-bromo-2-nitropropane-1,3-diol), aswell as 3-nitrobenzaldehyde working well when prepared ‘solvent-free’additionally or in substitution to solvent-based sample preparations. Ingeneral, the compounds for matrix are within the range between 50 and600.

Alternatively, the mass spectrum can be obtained using a API inlettypically used with ESI by maintaining the inlet below 150° C., andpreferably below 100° C. and placing the sample or matrix solution andthen analyte solution, or vice versa, onto, for example, filter paperand placing the sample on the filter paper substrate against the inletaperture. The paper creates sufficient vacuum for ionization to occureven though some air is able to flow through the gas permeable paper.MAIV matrices provide the first example of converting nonvolatilecompounds to gas phase ions by simply exposing matrix/analyte, thesample, to sub-atmospheric pressure. 6-Nitro-o-anisonitrile, andphthalic anhydride are examples of MAIV matrices that produce ions withvacuum assistance at ambient temperature additionally to those compoundsthat function as MAIV matrices on the vacuum source (listed in [00026]).2,5-DHAP is an example of compounds that are MAIV matrices when heat isapplied to the substrate or matrix.

Unlike MAII in which heat is applied to the inlet, in MAIV heat can beapplied directly to the matrix by, for example, heating the substrate towhich it is deposited. This is advantageous relative to MAII in that therequirement of a heated inlet to produce ions is eliminated so that awider range of instruments and conditions are useful with MAIV. Anycompound that produces gas-phase ions by exposure to sub-atmosphericpressure when cooled, at ambient temperature, or when the matrix isdirectly heated is considered a MAIV matrix. These matrix compoundssublime or evaporate when exposed to sub-atmospheric pressure at ambienttemperature or at temperatures below about 100° C. and spontaneouslyproduce ions from incorporated analyte for analysis by mass or ionmobility spectrometers. Because charged matrix:analyte particles ordroplets are believed to be the source of analyte ion formation in MAIV,desolvation of the charged particle/droplet can be enhanced with, forexample, application of heat, collisions of the chargedparticles/droplets with gas or a surface, and by fields such asradiofrequency fields used in MS.

In MAIV, ionization can be enhanced by shaking the substrate by, forexample, vibrations produced by an ultrasonic or a piezoelectric deviceor simply by a vortexer. Adding heat or cold to the surface on which thematrix is applied also can alter the ion abundance observed. Using thesemethods, the ionization event can be made to have a short duration withincreased ion abundance per unit time, or to be prolonged with less ionabundance over extended time. Ionization can also be prolonged bylimiting the area of the sample exposed to sub-ambient conditions suchas but not limited to the use the sample deposited inside of a capillarytube or pipet tip, made of, for example, glass, metal, or polymer,instead of being placed on a flat surface. Heating the matrix or thesurface onto which the sample is applied allows a wider range ofcompounds to perform as MAIV matrices. For example, 3-NBN producesabundant ions at sub-atmospheric pressure at substrate temperatures atleast as low as 25° C., whereas 2,5-DHAP produces analytical usefulresults when heat (>60° C.) is applied to the substrate.

MAIV is applicable with any instrument that has an ion source operatedunder vacuum conditions. Such instruments are commonly used with gaschromatography (GC)/MS and having electron and chemical ionizationsources, as well as with MALDI sources. With a vacuum source, the sampleconsisting of a MAIV matrix with incorporated analyte need only beexposed to the vacuum to begin producing ions. Heat applied to thesubstrate holding the sample under vacuum conditions is useful forenhancing ion formation with some matrices. Various methods for applyingenergy to aid desolvation of charged particles/droplets downstream fromthe surface, such as collisions, IR, microwaves, and radiofrequencyradiation, and other approaches known to those practiced in the art canbe used to increase analyte ion abundance. If the sample is placed closeto ion extraction and focusing lens, known to those practiced in theart, the analyte ions produced from the MAIV matrix will be acceleratedand guided to travel through the mass analyzer or ion mobility device tothe detector where a signal is generated for amplification andconversion using a computer to provide an output.

Alternatively, ion sources that are designed to work with atmosphericpressure ionization methods such as ESI, APCI, and atmospheric pressureMALDI also work with MAIV. There are a number of possibilities forproducing analyte ions for MS analysis using MAIV at atmosphericpressure. Simply exposing the MAIV matrix to the entrance aperture of anAPI inlet on a substrate creates sufficient vacuum for ionization tocommence. Holding the substrate, to which the sample is applied, ontothe inlet aperture of the mass or ion mobility analyzer produces ions bycreating a perfect or an imperfect vacuum seal at the entrance. Apermeable surface such as filter paper with the sample applied and driedcan be placed against the entrance, or a modified entrance having alarger aperture, to produce ions from larger sample surfaces or multiplesamples deposited (e.g., ideally using multichannel pipet dispenser) onthe substrate. Applying multiple samples to a strip of paper, orappropriate ribbon, and moving the strip or ribbon to successivelyexpose the samples to the vacuum produces a high throughput means ofanalyzing multiple samples individually. The samples not exposed to thevacuum may be at atmospheric pressure. Likewise, the sample can beapplied in a 2-dimensional array (e.g., a 96-well plate or pipet tiparray for loading of multiple samples simultaneously to the 2-Dsubstrate) and an x,y,(z)-stage employed to sequentially places thesamples at the entrance aperture of the mass or ion mobilityspectrometer. With API inlets, typically air flow is preferred so thatair permeable substrate or an imperfect seal provides optimum results. Asolution consisting of the matrix, analyte, and a volatile solvent such,but not limited to, methanol, acetonitrile, water, and mixtures thereofcan be injected into the channel to produce analyte ions. This method isalso subject to high throughput analyses.

With MAIV, the inlet linking atmospheric pressure and vacuum can havevarious size openings, including some larger than the pumping capacityof the instrument allows, so long as the substrate produces a sufficientvacuum seal not to vent the instrument. An isolation valve, as forexample a ball valve, can be used to isolate the vacuum of theinstrument from atmospheric pressure when the sample substrate is notagainst the inlet aperture.

One means of rapid and automated sampling is the use of well plates withthe samples in the wells. A well can be pushed against the inletaperture or the aperture of an extension of an inlet tube to create thesub-atmospheric pressure at the well bottom. The sample in the well isionized and swept into the analyzer for separation by mass-to-charge orsize, shape (also known as cross section), and charge. The well platecan be moved to expose the next well to the vacuum of the analyzerthrough the inlet. Turning off the vacuum isolation valve, which can bedone automatically, allows larger inner diameter inlet tubes to be movedto the next well position without venting the instrument. Larger inletinner diameter channels may improve the sensitivity of the analysis, butat the expense of requiring an in-line valve. A flat surface can beanalyzed in a similar fashion to the well plate by having the inletaperture with a sufficiently large opening to cover the entire sample.This can be done using, for example, a conical inlet.

Another alternative approach is to use, for example, a valve, such as aball valve, that has indentations to hold small containers in which thesample is placed. A container holding the sample can be loaded into oneof the indentations of the valve at, as a simple example, the upperposition of a four indentation valve. The upper indentation and lowerindentation are open to atmospheric pressure and the horizontalindentation opens to the analyzer vacuum. Turning the valve in onedirection moves the upper position with the container to the positionthat exposes it to the instrument vacuum. If a container is used to holdthe sample, the entrance to the vacuum in some embodiments is too smallfor the container to enter the opening. Ionization commences on exposureof the sample to sub-atmospheric conditions. In this position, the newupper indentation can be automatically loaded with the sample or blankor a container with a blank or the sample. Once sufficient ion currentis accumulated, a computer can send a signal to turn the valve so thatthe spent container is now in the bottom position and falls into a wastecollection and the second the sample is simultaneously exposed to vacuumto initiate ionization while the top position is again ready to beloaded with another the sample. Blanks can be run between the samples toeliminate potential carryover. Ions produced by such an arrangement canbe guided into the analyzer for analysis using a gas flow or using lenselements, including radiofrequency guides known to those practiced inthe art. Other arrangements which will be obvious to those practiced inthe art can be used with MAIV matrices to produce analyte ions for highthroughput with and without automation. Examples are tissue analysis,drug analysis, protein and peptide analysis, carbohydrate analysis, andin general small and large molecule analysis.

Besides the ease of ionization, low cost, and safety of MAIV and itsapplication to a wide array of instruments and approaches, the methodalso provides advantages for ionization where a low energy ionizationprocess is preferred. An example is the analysis of complexes, such asprotein-drug, or protein-protein complexes which are unstable to anyenergy source. Another application is in studying ion structures (crosssections) by ion mobility where energy supplied in the ionizationprocess can affect the ion structure. Another example is the ability toobserve structures directly from surfaces, as in biological tissue, tocharacterize the compounds in the tissue without application of energy,as happens with, for example, MALDI analysis. Yet another example is inthe analysis of analyte in thin layer chromatography (TLC) plates, paperchromatography, and 1-D and 2-D gel electrophoresis or from LC in anonline or offline approach. Analysis of blood spots directly from paperis yet another example. The substrate can be precoated with matrix sothat analyte only needs to be added. Such a simple and robust ionizationprocess, requiring only application of a matrix solution and exposure tosub-atmospheric pressure and capable of ionization of volatile (e.g.,drugs) and nonvolatile compounds (e.g., large proteins, fragilecomplexes or modifications), is expected to have numerous applicationsincluding in clinical analyses (e.g., high speed sample introduction ofbiological matrixes such as urine and whole blood), omics applications(using e.g., the more traditional bottom up approaches based on 1-D and2-D gels or LC or in top down proteomics), field portable instruments,and forensic analyses.

An additional advantage of this method relates to sensitivity.Ionization initiated through vacuum conditions is more sensitive thanionization initiated at atmospheric pressure because the transfer ofions from atmospheric pressure to vacuum is an inefficient process. Thisis directly seen with MALDI-TOF (a vacuum ionization method) instrumentswhich have comparable or better sensitivity than ESI (an atmosphericpressure ionization method) even though the efficiency of ionization isorders of magnitude less than the ionization efficiency of ESI. Thus,the MAIV method of ionization is highly sensitive because ions areformed under vacuum conditions eliminating the disadvantageous transferof ions from atmospheric pressure to the vacuum of the analyzer.

Another advantage of this method relates to continuous ion formation.One distinct advantage of this is that no hot/cold spot issues areobserved which limit MALDI applications. In MAIV, the sample can berapidly changed, contrary to the best efforts used with ESI and withoutcross contamination either through rapid moving surfaces (e.g., using an‘endless’ paper) or containers (e.g., using a ball valve arrangement).The continuous ion formation is also ideal for improved fragmentation todetermine the structure of an analyte using CID, ETD or ECD as examples.

A difficulty of the MAIV approach with ion sources built for vacuumionization is that MAIV matrices that do not require a heated substrateare volatile in vacuum and evaporation/sublimation begins as soon as thesample is placed under sub-atmospheric pressure conditions. The pumpdown time to reach a sufficiently low pressure for the mass spectrometerto operate can result in evaporation or sublimation of the sample in itsentirety if the matrix is too volatile. This can be circumvented to someextent by cooling the sample and/or the substrate before placing thesample on the substrate into the instrument. Another solution is to havea mechanically cooled substrate by, for example, refrigeration of thesubstrate or the substrate housing. Restricting the sample surface areaexposed to vacuum also extends the time for ion formation, but at theexpense of lower ion abundance during any single acquisition. Smallerpumping volumes can be engineered to allow fast pump down so that lessof the sample is lost before the instrument is ready to acquire data.

Another problem relates to the difficulty of introducing multiple thesamples into the vacuum for analysis. These problems are circumvented byhaving a system, where only one of the samples is exposed to vacuum atany one time. These approaches are also advantageous for quantitativeanalyses, especially incorporating internal standards. Finally,providing the sample substrate which can be heated increases the rangeof compounds that act as MAIV matrices spontaneously producing ions fromanalyte. The use of a laser below the threshold for ionization orablation of the matrix, or the employment of a gas flow, or gentlyrubbing the substrate exposed to sub-atmospheric pressure, or ultrasonicvibration of the container, or other means of shaking or vibrating (e.g.piezoelectric) the sample are means of initiating or enhancing MAIV.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates an embodiment of a system for introducing the matrixwith incorporated analyte, the sample, from a higher to a lower pressurefor ionization and analysis of the analyte by MAIV.

FIG. 2 illustrates another embodiment of a system for introducing thesample from a higher to a lower pressure for analysis using massspectrometry or ion mobility spectrometry.

FIG. 3 illustrates another embodiment of a system for introducingmultiple the samples individually from a higher pressure region to alower pressure region for analysis of the analyte incorporated in theMAIV matrix. It should be understood that the multiple samples can beanalyzed in rapid sequence or simultaneously through multiple channel.In dual channel mode, the second channel could realize the standardprepared with known concentrations as a MAIV sample.

FIG. 4 illustrates another embodiment of a system for automatically ormanually introducing the samples from a high pressure region, typicallyatmospheric pressure, to a sub-atmospheric pressure region forionization and transfer of the ions to an ion mobility or mass analyzerfor analysis.

FIG. 5 illustrates another embodiment of a system for automatically ormanually introducing the sample to sub-atmospheric pressure to initiateionization.

FIG. 6 illustrates another embodiment of the system represented in FIG.5.

FIG. 7 illustrates another embodiment of a system for introducing theMAIV matrix and analyte, the sample, in solution to a sub-atmosphericpressure region to initiate ionization.

FIG. 8 illustrates another embodiment of a system in which a laser isused to heat a small area of the sample to aid ionization by the MAIVmethod.

FIG. 9 illustrates the MAIV mass spectrum of bovine serum albumin (BSA)(Molecular weight [MW] ˜66000) using the vacuum MALDI source, hereafterthe vacuum source, of a Waters SYNAPT G2 ion mobility spectrometry (IMS)mass spectrometry (MS) instrument without the laser and using 3-NBN asthe matrix.

FIG. 10 (top) illustrates MAIV of lysozyme (MW 14300) and (bottom) itscomplex with a small peptide (PNAP) analyzed from buffered solutionusing 3-NBN as matrix on the vacuum source.

FIG. 11 illustrates MAIV of the fragile non volatile compounds: (top)GT1 b gangliosides (MW 2128 and 2156) and (bottom) a phosphorylatedpeptide (MW 1332) using the vacuum source and negative mode detection.

FIG. 12 illustrates MAIV of the membrane protein bacteriorhodopsin (MW27000) 3-NBN as matrix using the vacuum source. Multiply charged ionsare observed (top).

FIG. 13 illustrates MAIV of a variety of analytes using the vacuumsource.

FIG. 14 illustrates MAIV using the vacuum source with 3-NBN as thematrix and 50 fmol of ubiquitin. The inset illustrates the isotopicdistribution of charge state +10 of ubiquitin.

FIG. 15 illustrates analyses of 5 femtomoles of clozapine. The leftpanel is the MAIV results using 3-NBN as matrix on the vacuum source andthe right panel using ESI.

FIG. 16 illustrates MAIV using 3-NBN as matrix on the vacuum sourceusing a capillary as the substrate into which the sample was dried toprolong ionization.

FIG. 17 illustrates another method for extending the time of ionizationusing 3-NBN with vacuum source MAIV.

FIG. 18 illustrates MAIV using the vacuum source and the substrate at−80° C. to obtain the MS/MS spectrum of clozapine (MW 328) at 900attomoles deposited on the metal plate.

FIG. 19 illustrates MAIV mass spectra using the ESI source and thematrix 3-NBN and paper chromatography to separate compounds in a markerpen.

FIG. 20 illustrates MAIV from (top) a thin layer chromatography (TLC)plate and (bottom) a metal foil using the vacuum source and 3-NBN asmatrix.

FIG. 21 illustrates MAIV of a peptide using 3-NBN as matrix and thevacuum source.

FIG. 22 illustrates MAIV applying the matrix 3-NBN to a small area of a20 dollar bill using the vacuum source.

FIG. 23 illustrates MAIV using the vacuum source and 3-NBN as matrix.Ions are instantaneously formed and separated as well as detected(IMS-MS).

FIG. 24 illustrates the MAIV mass spectra of a delipified tissue sectionusing the 3-NBN matrix and the vacuum source.

FIG. 25 illustrates MAIV of drug dosed urine characterizing Cetirizine(MW 388) using the vacuum source and 3-NBN as the MAIV matrix (metalplate substrate).

FIG. 26 illustrates MAIV of 1-D gel (gel slices analyzed after gelelectrophoresis) using the vacuum source.

FIG. 27 illustrates MAIV using the vacuum source of (top) a syntheticpolymer (PEGDME 2000) using LiCl and (bottom) ubiquitin (MW 8561) and 1M NaCl as salt additives to the sample solution using 3-NBN as thematrix.

FIG. 28 illustrates the MAIV analyses of (top) a wet sample spot and(bottom) completely dried sample spot using 3-NBN as the matrix prior tointroduction to the vacuum source.

FIG. 29 illustrates eight MAIV matrices using the vacuum source. Thematrix structure used is indicated in each mass spectrum.

FIG. 30 illustrates MAIV of lysozyme (MW 14300) using a binary matrixmixture of 3-NBN:CHCA 1.6:1 and (top) the vacuum source or (bottom) theAP source.

FIG. 31 illustrates MAIV of whole blood extracted from a band aid usingthe modified ESI skimmer cone (from here on referred to as the APsource).

FIG. 32 illustrates using the source displayed in FIG. 31 to analyze byMAIV-IMS-MS a mixture of five drugs.

FIG. 33 illustrates MAIV of 40 fmol clozapine and 20 pmol bovine insulinusing 3-NBN. Incorporating the IMS dimension increases the dynamic rangeof the experiment.

FIG. 34 illustrates MAIV of nicotine and nornicotine using 3-NBN.Incorporating the IMS dimension separates isobaric molecules ofnornicotine and 3-NBN.

FIG. 35 illustrates MAIV of lysozyme using 3-NBN from a bufferedsolution showing the mass spectra (top) in which the solution of analytewas added to filter paper and while wet the matrix in a separatesolution was added, and (bottom) the matrix solution was added to filterpaper and dried. To this precoated matrix layer the analyte was added.

FIG. 36 illustrates MAIV using 3-NBN of four different samples in lessthan 1 minute using the AP source sliding the paper strip, onto whichthe sample was applied, across the inlet opening to createsub-atmospheric pressure at the sample.

FIG. 37 illustrates MAIV using (top) a pipet tip as a sample substrateas demonstrated in FIG. 7.

FIG. 38 illustrates MAIV with 3-NBN as the matrix using a 8-channelmultiple pipet tips dispenser.

FIG. 39 illustrates MAIV and the 3-NBN matrix of LSD (MW 323) using theAP source with a nozzle extension analyzing the sample from the bottomof one well of the well plate similar to FIG. 6.

FIG. 40 illustrates MAIV and the 3-NBN matrix of nicotine (MW 162) usingthe AP source analyzing the sample from well of the 384-well plate(pictures top left).

FIG. 41 illustrates MAIV analyses of Clarithromycin (MW 747) directlyfrom a tablet using the 3-NBN matrix and the AP source.

FIG. 42 illustrates MAIV using the AP source and 3-NBN as the matrix.Top: Picture and TIC provide view of the set up and the TIC's (right)provide an indication of the duration of the ionization process.

FIG. 43 illustrates MAIV-ETD using the AP source.

FIG. 44 illustrates MAIV using 3-NBN of five different samples in lessthan 3 seconds using a gently adjusted laser to heat the sample close tothe skimmer cone similar to FIG. 8.

FIG. 45 illustrates nine MAIV matrices using the AP source. Thestructure and temperature used is indicated in each mass spectrum.

FIG. 46 illustrates a MAIV matrix/temperature study (in the range of 30°C. to 450° C.) using the 3-NBN, 2,5-DHAP, 2-NPG,methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, and6-nitro-o-anisonitrile matrixes and the AP source of the SYNAPT G2(skimmer cone source, maximum temperature that can be applied is 150°C.) and the LTQ Velos (inlet tube, maximum temperature that can beapplied is 500° C.).

FIG. 47 illustrates MAIV-IMS-MS: (right panel) 2-D plot display of drifttime vs. m/z and (left panel) mass spectra of beta-amyloid (1-42) (MW4511 Da) using MAIV with 3-NBN matrix in (I) positive mode and (II)negative mode on the intermediate pressure vacuum source of SYNAPT G2,and (III) ESI comparison in negative mode.

DETAILED DESCRIPTION OF DISCLOSURE

In the invention described here, ionization occurs in thesub-atmospheric pressure that can be associated with the ionizationregion of commercial mass spectrometers such as the commercial ormodified inlets (inlet tubes, skimmers, etc.) of an atmospheric pressureionization source or a vacuum ionization source of a mass spectrometeror ion mobility spectrometer facilitated by an appropriate smallmolecule matrix such as, but not limited to 3-nitrobenzonitrile (3-NBN),2-nitrobenzonitrile (2-NBN), 5-methyl-2-nitrobenzonitrile, coumarin,methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, bronopol(2-bromo-2-nitropropane-1,3-diol), 3-nitrobenzaldehyde,6-nitro-o-anisonitrile, and phthalic anhydride and certain derivativesthereof. More compounds such as 2,5-DHAP act as MAIV matrices when thesample or the sample substrate are heated. Other means of creatingsub-atmospheric pressure to the sample can be used with this method suchas a pin-hole leak. Providing means for enhanced evaporation orsublimation of matrix from the gas phase charged particles or dropletsproduced in the initial spontaneous ionization event can enhance ionformation. Means of providing energy to enhance this desolvation processinclude heat by such means as radiative as in microwave, IR, visible, orUV radiation, radio frequency fields, gas flow and collisions withgaseous molecules or solid surfaces, and other methods known to thosepracticed in the art. None of the matrices require a force to propel thematrix:analyte association into enter the gas phase.

Multiply and singly charged ions of volatile and nonvolatile analytecompounds are formed without the need for application of voltages foranalyte ionization; the commercial mass spectrometer separates anddetects the formed ions according to the mass-to-charge ratio and ionmobility analyzers by the charge, size, and shape of the ion. No lasersare needed, but can be used to provide heat to the sample surface or thesubstrate onto which the sample is applied to enhance spontaneousionization and increase the compound types that work as MAIV matrices.Using a laser beam to heat the matrix provides identical results toheating the sample by, for example, heating the substrate. A laser beamprovides a means of locally heating a specific area of the sample.Similarly, a gas flow can be used to evaporate the sample gently. Justas heating the sample can extend the compound types that work as MAIVmatrices spontaneously producing ions using exposure to sub-atmosphericpressure, preferably between 750 mmHg and 1×10⁻⁷ mmHg, cooling thesample can also extend the method to more volatile compounds acting asMAIV matrices.

The matrix may be mixed with the analyte in a solvent and the sampleapplied to a substrate for placement at or near the ionization region(e.g., inlet aperture of the atmospheric pressure or the ion extractionelement of the intermediate pressure mass spectrometer or ion mobilityspectrometer) so long as the sample is at sub-atmospheric pressure.Depending on the MAIV matrix, heat may be applied to the matrix or thesubstrate onto which it is applied and to the inlet region of the massspectrometer. The solvents can be the same or different for the matrixand analyte, and the analyte can be in the same solution as the matrixor separate solutions that are mixed before analysis. Salt additives canbe used for certain compounds such as but not restricted to syntheticpolymers. Buffer conditions can be used for protein-small ligand andprotein-protein complexes but are not restricted to these examples.Ammonium salts can be added to the matrix or analyte to reduce chemicalbackground or enhance analyte ion abundance. MAIV operates at low andhigh pH using a variety of different acids or bases, and at neutral pH.The sample solution can be dried or in solution when introduced againstor within a region near the inlet influenced by the low pressure of theinstrument. If the sample is introduced in solution the lower pressureconditions and heat aids solvent vaporization and subsequentlyionization. Negative and positive mode measurements can be performedaccording to the preferred analyte structure such that acidic molecules(e.g., fatty acids) preferentially ionize in the negative mode and basiccompounds (e.g., hydrophilic peptides, drugs) preferentially ionize inthe positive mode. With the MAIV method, the mass range is extended bymultiple charging of the analyte for use of low and high performancemass spectrometers with their full capabilities including, but notlimited to, accurate mass measurements, high mass resolutionmeasurements, improved ion mobility separation, high efficiencydetection using image charge detection, high efficiency fragmentation ofmultiply charged ions using CID, and high performance fragmentationusing ECD and ETD known to those practiced in the art. Small moleculessuch as drugs, prescription and illicit, and their metabolites, lipids,carbohydrates, triglycerides, proteins and protein-complexes are someanalyte examples. Because hot/cold spot issues, limiting MALDI atatmospheric pressure and vacuum, are eliminated and ion production iscontinuous as in ESI, irreproducibility and quantitation issues areminimized with MAIV, similarly to the continuous ion formation in ESI.

Multiplexing with MAIV exceeds the throughput of direct injection ESIand does not have issues with clogging capillary tubes or the need ofheated desolvation gas. In MAIV, less chemical background is observedrelative to ESI and MALDI. MAIV is also tolerant to the presence ofsalts commonly found in biological materials (urine, tissue, whole bloodas examples). This method performs well at and near physiologicalconditions without the need of addition of acid or base, extending theapplicability to acid and base labile analytes and chemical andposttranslational modifications.

Cooling of the sample substrate extends the life time of the ionformation process and the applicability to a number of matrices. Thesample container outlet to vacuum conditions can be restricted extendingthe life time of the MAIV ion formation process and a means of cappingthe containers until ready for ionization offers the ability tomultiplex many the samples in a vacuum source similar to vacuum MALDIbut without the need of the laser used with MALDI. In case of a wellplate array of samples, additionally or alternatively, a film(inexpensive and disposable) can be used to cover the array well plateopenings that can be penetrated individually to initiate MAIV ionizationand analyses. The lifetime of the signal can also be extended by use ofbinary matrices for which one of the matrices is a common LSI or MALDImatrix. Examples are adding 2,5-DHB, α-cyano-4-hydroxycinnamic acid(CHCA), or sinapinic acid (SA) to a MAIV matrix in which the MAIV matrixcan be as low as 5% of the mixture. By extension, more than two matricescan be mixed so long as one is a MAIV matrix.

Because no additional energy or force is required, MAIV is applicable tofragile molecules such as gangliosides and noncovalent complexes. Gasflow aids in more rapid ionization of the sample provided thatsub-atmospheric pressure conditions are maintained and also extends theapplicability to more matrices. Gases, collisions with surfaces, andradiofrequency fields downstream from the sample aid in producing higherabundance of the analyte ions, especially with less volatile MAIVmatrices and two or more matrices combined with one being a MAIV matrix.

Specificity of analyte ionization can be obtained through suitablematrices and matrix combinations. Temporal resolution is achieved usingfast introduction of the sample to the vacuum of the mass spectrometerinlet. Spatial resolution analyses is obtained by miniaturizing thematrix deposition area onto the surface of interested, or by using apin-hole leak to provide vacuum conditions to a small area of thesample, or a gas flow pointed at the surface or by use of a laser belowthe matrix/laser threshold to heat a small area of the sample in fluidcontact with the sub-atmospheric pressure of the analyzer.

The substrate onto which the matrix is placed may be a variety ofmaterials that do not provide chemical contaminants that unduly add tothe background or suppress ionization. The substrate may be imperviousto surrounding atmosphere, typically air or nitrogen gas, or permeableallowing some flow of gas through the substrate. Typically, with anatmospheric pressure ion source, gas flow aids transfer of ions to themass analyzer, and thus, some airflow enhances ionization using inletsof API sources. The gas flow can be achieved by use of permeablesubstrates, imperfect vacuum seals, or by controlled gas leaks. Vacuumionization sources use voltage to direct ions from the substrate towardsthe analyzer. Materials for the substrate such as, but not limited to,paper (especially filter paper), metal (plate, foil, or mesh), glassplates or tubes (filled/deposited inside or outside with the sample),synthetic or natural polymers (e.g., well plates, pipet tips), andfibers can be used to introduce the sample to the sub-atmosphericpressure region where ionization commences. Substrates can be materialsused for 1- and 2-dimensional separations (paper chromatography, TLCplates, gels (e.g., SDS-PAGE, agarose). The MAIV matrix is depositedmanually or sprayed on the surface or deposited directly into thematerial containing the analyte, the surface affixed to or exposed tothe vacuum for MAIV ionization of individual spots, regions or theentire 2-D and 3-D surface of flat or curved objects. Sampling thesurface in a systematic fashion, similar to imaging by MS, provides thelocation of the respective analyte such as a small drug from a dollarbill or a protein from a 1-D gel but without the necessity of a laserwhile providing spatial and temporal analyses. The analyte to beanalyzed can be on a material (e.g., pesticide on a fruit) or part of amaterial (e.g., active ingredient of a drug directly from a pill(tablet) surface, illicit drugs in hair, mouse brain tissue, living skinor flesh). Some MAIV matrices are natural content of spices (e.g.,coumarin) or part of cosmetics (e.g., bronopol).

With MAIV, a variety of means can be employed to achieve rapid andautomated analyses. For example, a system can be employed using a metal,polymer, paper, cotton, or fiber ribbon onto which matrix and analytecan be applied by syringe or automatically using an autosampler known tothose practiced in the art. The ribbon may be pulled from a roll onto asecond roll so that matrix and analyte can be applied separately ortogether onto the ribbon automatically or by hand, using for example asyringe, placed between the first roll and the instrument inlet. Thesecond roll can be used to pull the ribbon so that it unwinds from thefirst roll moving the sample across the inlet, exposing the sampledeposited continuously or discontinuously, to the vacuum of the massspectrometer. In this way, multiple analytes are analyzed sequentiallyas the samples cross the sub-atmospheric region at the mass spectrometeror ion mobility spectrometer inlet aperture. It should be noted thatrolls of ribbon are not required nor is it necessary to use a ribbon, aseven sheets of, for example, filter paper with the sample applied in a2-D grid pattern can also be automatically moved, irrespective of thedirection and at a desired speed, across the inlet entrance as describedabove to effect ionization of analyte. In some arrangements, thesubstrate surface does not have to be in fluid contact with the inletaperture but sufficiently close to experience the sub-atmosphericpressure in close proximity to the inlet aperture. The gas flowing intothe inlet and across the sample, whether by passing through a permeablesubstrate or by way of a poor vacuum seal of the substrate with theinlet aperture, can be warmed to aid the initial ionization process.Pipet tips (single, or arranged in a line or array) dried, semi-dry, orwet with the sample can be brought near or in fluid contact with theinlet aperture and a robot can be used to automate this process. Againthe ionization process is initiated when the sample experiencessub-atmospheric pressure. Similarly, tissue sections and gel samplesurfaces can be specifically analyzed using this principle by using axyz-stage to bring a specific sample area to close proximity or bypenetrating the surface layer. Well plates, commonly used for highthroughput studies, will also suffice as a substrate. The sample loadedinto wells can be allowed to dry and use of a robot or x,y,(z)-stage canmove the various wells individually to the ionization region. The massspectrometer or vacuum ion mobility spectrometer's inlet can be extendedwith a tube rather than the necessity of bringing the well plate orother surfaces that may be flat to the inlet of the instrument of amodified API inlet as long as the extension head is provided to hold asufficiently wide area and provide a good enough vacuum seal. Either thewell plate can be moved or the inlet tube extension moved so as to havethe sample in a well in intimate contact with the inlet or inletextension. Using, for example, the MAIV matrix 3-NBN eliminatesinstrument contamination by the matrix because the matrix readilysublimes through exposure to the vacuum of the instrument. The matrixcan be pre-applied to a surface such as a ribbon, paper, or well platein order to simplify the process of loading the sample. With pre-loadedmatrix, only a solution of analyte needs to be applied. Analyteconcentrations from femtomolar to millimolar can be used routinely withthe MAIV method. Cooling the substrate to −80° C. before insertion intothe vacuum source area extends the sensitivity into the mid to highattomole range by delaying ionization until the sample is loaded intothe proper position for ion transmission.

Application of matrix to a surface using methods for matrix depositionin MALDI and LSI imaging allows images of certain compounds includingbut not limited to drugs, metabolites, lipids, peptides and proteinsadministered and endogenous, from chemical surfaces such as catalystsand synthetic polymers, to be obtained using MAIV methods. Examplesurfaces include films, cell cultures, microbial communities, or tissuesections from certain organs or flesh. For example, moving the surfacecovered with the MAIV matrix across the inlet aperture to the vacuumregion of the instrument produces a low resolution image without theneed of a laser. The resolution of the image is related to the diameterof the area exposed to the vacuum by the inlet aperture. Thus, apin-hole leak will produce a higher resolution image but with lowersensitivity than a larger aperture. Analyses of the composition andcomposition changes relative to position can be obtained for the entiresurface or specifically targeted areas of interest. Simply depositingmatrix in a small area of the surface and exposing the surface tosub-atmospheric pressure conditions produces ions only from compounds atthe surface exposed to the matrix solution. A resistive heater or gasstream can also be used to locally heat a specific area or array of asurface. A laser can also be employed similar to LSII using a MAIVmatrix, but unlike LSII, the laser is used to heat the sample and notfor ablation. Mass spectrometers without a heated inlet linkingatmospheric pressure and the vacuum of the mass spectrometer can be usedfor imaging with high spatial resolution using a MAIV matrix and alaser. In this case, the sample may be manipulated at atmosphericpressure or higher pressure, and unlike LSII and MAII, the inlet can beat or near ambient temperature. Alternatively, if the sample issubjected to sub-atmospheric pressure, the laser can be used simply toheat the surface to effect ionization. In this case, infrared lasers arepreferred with matrices that do not absorb at the ultraviolet orvisible. For imaging, MAIV matrices that require the sample or substratebe heated before spontaneous ionization commences are preferred.

A MAIV matrix in solution using volatile solvents including water whenintroduced into an inlet tube to the vacuum of a mass spectrometersubstantially lowers the temperature required on the inlet tube relativeto SAII. By using a Tee adapter, matrix solution can be added to, forexample, a LC effluent to enhance ion abundance and the multiple chargestates allowing extension of the mass range of analytes such as proteinsand large peptides to high performance mass spectrometers with massrange limited capabilities and to enhance fragmentation using CID, ETD,and ECD and variations thereof as well as increase the effectiveness ofion mobility separations on mass and ion mobility spectrometers lackingthe application of an heated inlet tube source. The sample can besupplied indirectly using methods common to offline LC-MALDI (solidstate from e.g., surfaces or well plates) or online LC-ESI (solutionstate, from nano and microliter flow) known to those practiced in theart.

Some MAIV matrices (e.g., 3-NBN) can not only produce ions for analysis,but can also be used as an ETD reagent. Thus, a glow discharge is usedfor producing negative ions of gaseous matrix molecules within thevacuum of the mass spectrometer, usually the first vacuum stagefollowing the inlet. The negative ions of the matrix can be trapped byion optics such as quadrupole elements within the mass spectrometer andallowed to react with positive ions produced from the analyte in thematrix trapped in the same region. Electron transfer from the negativeions of the ETD reagent to the multiply charged positive analyte ions inthe trap cause ETD fragmentation of the positive ions for furtheranalysis which will be obvious to those practiced in the art. Thecharged ETD reagent gas can be used on mass spectrometers such as theWaters Corporation SYNAPT G2 to perform sequence and posttranslationalmodification analysis of peptides and proteins. Analyte fragmentation isachieved by switching between cation and reagent anion injection into atrap region of the mass spectrometer; this process can be automated andaccomplished within a few hundred milliseconds. The use of this methodrelative to commonly used ETD methods is that the MAIV matrix is alsothe ETD reagent eliminating the necessity for a separate apparatus tointroduce the ETD reagent. ETD is applicable to vacuum sources (MALDI,EI, CI) through modifications obvious to those trained in the art.

Ion mobility and mass spectrometers with and without coupling the twotechnologies can be employed with this analyte ionization method such asthose used for clinical, homeland security, field portable and hand-helddevice applications.

FIG. 1 is a representation of one embodiment of a device for introducingthe sample to a region of sub-atmospheric pressure where gas phase ionsfrom the analyte are produced without the necessity of a laser, voltage,or any external energy or force applied to the sample of substrate,although energy sources may be useful for certain applications and foruse of certain MAIV matrices. In the embodiment shown, a valve (23)separates a higher pressure region (17) from a lower pressure region(10). The higher pressure region 17 may be at atmospheric pressure andthe lower pressure region 10 at an intermediate pressure that liesbetween the higher pressure in region 17 and the vacuum of the analyzer.The valve in one representation is a ball valve with an opening 32through the ball that, when opening 32 is in the direction shown in thefigure, segregates the higher (17) and lower (10) pressure regions sothat they are no longer in fluid contact with each other. When probe 21is inserted into the valve body 18 so that the sample holder 22 insertsinto the valve body 18 through channel 19, it provides a sufficient sealso that valve 23 may be turned 90 degrees so that the opening 32 placesthe sample holder 22 in fluid communication with the lower pressureregion 10 without detrimentally raising the pressure in region 10.Turning ball valve 24 so that opening 32 is 90 degrees to the flow ofgas from region 17 to region 10, allows removal of probe 22 by blockingthe flow of gas without substantially affecting the pressure in region10. Placing the sample 29 into the substrate 25 and placing thesubstrate 25 into the substrate holder 22 allows the sample 29 insubstrate 25 to be placed into channel 19 using probe 21. Ions from theanalyte are formed from the sample when valve 23 is turned 90 degrees sothat opening 32 of the valve is aligned so that the sample 29 is influid contact with the sub-atmospheric pressure of region 10. Othervalve technology that separates a lower from a higher pressure regionmay be used. The analyte ions produced from sample 29 on being exposedto sub-atmospheric pressure are extracted through opening 32 andaccelerated and guided into the mass analyzer using lens elementsrepresented by 25 and know to those practiced in the art. The small gasvolume created when probe 21 blocks opening 19 allows valve 23 to beopened without having detrimental impact on the pressure in region 10.In other representations, opening 19 can be larger and the pressurereduced in this region by a separate pumping stage that can be openedand closed by an operator or automatically. The method of sampleintroduction to vacuum can be manual or automated. By using this methodof the sample 29 introduction directly into a vacuum region of a massspectrometer, ion losses associated with transfer of ions fromatmospheric pressure to vacuum are eliminated and the sensitivity of theanalysis improved. Sensitivity is also improved using this introductionmethod because the time that the sample experiences sub-atmosphericpressure to the time analysis begins is short.

FIG. 2 is a representation of another embodiment of a device forintroducing the sample to a sub-atmospheric pressure region associatedwith a mass spectrometer or ion mobility spectrometer for analysis ofgas phase ions spontaneously formed from the analyte in the sample whenexposed to sub-atmospheric pressure. In this embodiment, 24 represents avacuum valve within a casing, the casing or body of the valve is notshown for simplicity. The valve can turn as shown by 13 either manuallyor automatically. In this representation, the valve has four wells 22that hold substrate 14 into which the sample 29 is placed. It isunderstood that more or less than four wells may be used. The sample mayalso be placed directly into the wells rather than onto or into asubstrate 14. The valve isolates a higher pressure region 17 from thelower pressure region 10 that is in fluid contact with the vacuum of theanalyzer. In this representation, substrate 14 containing the sample 29is placed in the top well as denoted by the top arrow in the drawingpointed downward. It is understood that other representations not shownare also operational. Turning the valve clockwise, as shown by 13, by 90degrees places the substrate 14 and sample 29 in fluid contact with thelower pressure region 10. In the embodiment, restriction 36 prevents thesubstrate from exiting the valve well 22. Once exposed to thesub-atmospheric pressure of region 10, ionization of the analytecommences. The analyte ions, which may be singly or multiply charged,are extracted and guided through lens elements 25 into the analyzer forseparation by mass-to-charge (m/z) and fragmentation using massspectrometry or separation by charge, shape, and size by ion mobility.As shown in the illustration by the lower arrow pointing downward, thesubstrate that had previously been exposed to vacuum, after turning thevalve 90 degrees is in the lower position and falls from well 22 ofvalve 24 into waste container 33. The empty valve can be rotated to thetop position to be filled with another the sample 29. Blanks may be runbetween samples to assure no carryover between samples. If substrate 14is not used and the sample 29 is placed directly into well 22, the well22 to the left and open to region 17 in this embodiment is cleaned whilethe well to the right in the representation is exposed to vacuum ofregion 10 of ionization of the sample in that well. This process can bemanual or automated.

FIG. 3 is an embodiment of another representation of an arrangementwhereby multiple samples can be acquired sequentially using a vacuumsource 55 of a mass spectrometer with a vacuum stage entrance 50 similarto those used for introducing the sample stage in MALDI. Thisarrangement, known to those practiced in the art allows stage 56 to beinserted into the vacuum source chamber 55 and held against the plate 57that separates region 55 form region 10. Stage 56 fits tightly againstplate 57 and may be spring loaded to form a vacuum seal except for asingle channel 19 in plate 57 leading to the vacuum region 10 in fluidcontact with the lower pressure region of the mass analyzer. Ionextraction lens 25 is directly aligned with channel 19 to extract ionsformed from the sample 29 to be into region 10 to be separated bymass-to-charge in the mass analyzer. Chamber 56 can be moved withx,y,z-stage 51 to expose different wells 22 to channel 19 and the vacuumof region 10. In order for each well 22 in stage 56 to be atsufficiently high pressure to minimize sublimation or evaporation ofsample 29, the stage is sealed from the vacuum in the source 55 so thatthe pressure in 56 can be higher than the pressure in region 55. A tube53 leading from region 17 that may be at atmospheric pressure allows gasto flow into stage 56 controlled by a leak valve 60. The leak iscontrolled by a valve so that the pressure in 56 is regulated. Acapillary channel 61 between stage 56 and well 22 allows sample 29 to bein fluid contact with the pressure of stage 56. Heater 39 in someembodiments is used to heat stage 56 and the sample 29. Using a MALDIsample plate and a MALDI source only a single sample can be loaded intothe vacuum for ionization. In this arrangement multiple samples can beloaded into the vacuum source 55 and sealed from the vacuum so thationization does not occur until the sample 29 is exposed to the vacuumof region 10 by being aligned with channel 19. Each well 22 is separatedfrom every other well. The advantage of exposing the sample tosub-atmospheric pressure of a vacuum ion source is that transferring theions to the analyzer is more efficient than with atmospheric pressureion sources. Other embodiments of such an arrangement can be envisionedincluding an arrangement in which the wells are sufficiently large tohold a section of tissue for analysis by application of a MAIV matrix tothe tissue and inserting the stage with tissue into the vacuum source.

FIG. 4 is a representation of an embodiment of a method that can beutilized to apply the sample to a substrate, which in some embodimentsis a belt or ribbon. The substrate 14 transfers the sample 29 applied tothe substrate 14 by device 20 to a sub-atmospheric pressure region 19for ionization of the analyte and transfer of the ions into a mass orion mobility analyzer for analysis of the analyte. In this embodiment, ahigher pressure region 17, which in some embodiments is at atmosphericpressure, is in fluid contact with a lower pressure region, which insome embodiments is the first vacuum stage of an analyzer, by an inlet18 with channel 19. The channel 19 of inlet 18 provides fluid contact ofregions 17 and 10 and in some embodiments is the inlet of an atmosphericpressure ionization mass spectrometer or ion mobility spectrometer andin some embodiment is a skimmer cone or a tube inlet. In someembodiments the channel of inlet 18 is sufficiently large that ittransmits more gas from region 17 to region 10 than the pumping systemof the ion mobility or mass analyzer can tolerate when substrate 14 isnot covering channel 19 of inlet 18. In such an embodiment, a valve canbe inserted into inlet 18 to restrict gas flow when substrate 14 is notcovering channel 19. Alternatively, the channel 19 of inlet 18 can bemade sufficiently small that the analyzer is operational when the inletis not covered by the substrate 14 as is common with inlets ofatmospheric pressure mass spectrometers.

In one embodiment of the method describe here, a spindle 11 holds asubstrate 14, which is one embodiments is a ribbon. The substrate 14 insome embodiments is made from paper, especially filter paper, fabric,polymer, plastic, metal, or other material that does not contributesubstantially to the ions observed by the analyzer. The substrate 14 ispulled over rollers 15 and onto spindle 12. The spindle 11 in someembodiments is spring loaded to keep substrate 14 tight between spindles11 and 12. Spindle 12 turns in direction 13 to pull the substrate 14from spindle 11 over rollers 15 and onto spindle 12. Axle 16 in someembodiments is attached to a motor drive to turn spindle 12. Device 20,which in some embodiments is a pipette tip, loads the sample onto ribbon14 while the ribbon is moving from spindle 11 to spindle 12. In anotherembodiment device 20 is an autosampler known to those practiced in theart and is used to introduce analyte in solution to the substrate 14. Insuch an arrangement, a second device 20, either before or after theautosampler, applies the MAIV matrix in a solvent to the substrate 14using, for example, a syringe pump. The entire assembly is positioned sothat the substrate 14 moves across channel 19 of inlet 18 so that thesubstrate 14 is either in direct contact with channel 19 of inlet 18 orin very close proximity to channel 19 of inlet 18. The sample applied tosubstrate 14 from a solution by device 20 dries as the sample movestoward inlet 18. When the sample experiences the vacuum at channel 19,ions are formed from the analyte in the sample 29. In thisrepresentation, substrate 14 is gas permeable. A heated gas flow (notshown) is directed in a narrow diameter stream at substrate 14 in closeproximity to the inlet channel 19 of inlet 18. By heating the substrate14 or the sample 29, ionization may be enhanced. In one embodiment,inlet 18 is heated to enhance evaporation of sublimation of the matrix.The movement of the substrate 14 and application of the sample 29 to theribbon in one embodiment is automated.

FIG. 5 is a representation of an embodiment of another method forobtaining rapid and automated analyses of the sample using the MAIVmethod. Inlet 18 with opening 19 provides fluid communication between ahigher pressure region 17 and a lower pressure region 10. The higherpressure region in some embodiments is at atmospheric pressure and thelower pressure region 10 is in fluid communication with the vacuum ofthe mass analyzer or ion mobility analyzer. Plate 27 has a frame 27 athat holds a mesh or screen 28 which is gas permeable. Substrate 14 inone embodiment is filter paper onto which the sample 29 is loaded,typically using 1 or a few microliters of the sample 29 in a solvent.Substrate 14 is affixed to plate 27 so that it partially or entirelycovers screen or mesh 28. The assembled device is attached to a stagethat moves plate 27 with substrate 14 held close to or in intermentcontact with channel 19 of inlet 18 in two or three directions as iscommon using xy- and xyz-stages used with microscopy. The plate 27 withsubstrate 14, having the samples 29 applied, is placed perpendicular toinlet 18 and either touching inlet 18 so as to cover opening 19 or verynearly touching inlet 18. In the case where the substrate 14 does notphysically touch inlet 18, it must be sufficiently close to the entranceto channel 19 to create a sub-atmospheric pressure environment for thesample 19 when it and inlet 19 are close proximity with one another.When the sample 19 experiences a lower pressure than in region 17, dueto the influence of region 10 through inlet opening 19, the sample 29produces gas phase ions from the analyte for analysis in the analyzer.Plate 27 can be moved using an xy- or xyz-stage 26 to sequentiallyexpose each the samples 29 to sub-atmospheric pressure to produceanalyte ions for analysis. This process in some embodiments is automatedsimilar to matrix assisted laser desorption, but here the plate 27,substrate 14, and the sample 29 in some embodiments is at atmosphericpressure rather than vacuum. Gas blown through a heated tube (not shown)of diameter similar to the diameter of the sample spots applied to thesubstrate can be used to enhance ionization with some matrix materials.Likewise, heating inlet 18 can enhance the MAIV process when usingcertain MAIV matrices.

FIG. 6 is yet another representation of an embodiment of a method tointroduce the sample 29 to sub-atmospheric pressure from a high pressureregion 17, typically at atmospheric pressure for ionization of theanalyte and analysis by mass spectrometry or ion mobility spectrometry.In this embodiment, inlet 18 with channel 19 provide fluid communicationbetween a higher pressure region 17 and a lower pressure region 10similar to FIG. 5. The inlet 18 has an extension 28 which extendschannel 19 so that it flares into a cone with a larger opening to region17. Substrate 14 with the sample 29 is manipulated using x,y-stage 26 tosequentially move the samples 29 so that they are in intermit contactwith channel 19 of extension 28. In one embodiment, the sample 29 isplaced in wells 22 in substrate 14. The exit of channel 19 to region 17has a larger diameter than the diameter of the wells 22 of the substrate14 so that the wells 22 and the sample 29 are in fluid contact with thelow pressure region 10 of an analyzer when the wells 22 and the channel19 are perfectly aligned so that the sample 29 is at sub-atmosphericpressure and analyte ions are formed. The gas leak through the well 22when not precisely aligned with channel 19 aids in transferring theanalyte ions into the analyzer for analysis. This method is someembodiments is automated and controlled by a computer.

FIG. 7 is another representation of an embodiment of a method forionizing the sample using the MAIV method. In this representation, thesample 29 composed of a MAIV matrix and analyte in solution isintroduced directly into opening 19 of inlet 18 using, for example, apipette 20 with solution 31 containing the sample 29. Addition of a MAIVmatrix to the solution substantially reduces the temperature requiredfor inlet 18 to produce ions of the analyte relative to solvent assistedinlet ionization, SAII, which uses a solvent without added matrix. Thus,abundant ions can be produced from both skimmer and tube inlets below150° C., a temperature in which SAII does not produce ions in a skimmerinlet and produces analyte ions in low abundance in a tube inlet. Insome embodiments, this method is automated.

FIG. 8 is another representation of an embodiment demonstrating how theMAIV ionization method can be used in conjunction with a laser. Althoughthe representation is of an atmospheric pressure ionization inlet, thisapproach also is operational with a vacuum source. Shown in theembodiment in FIG. 8 is a higher pressure region 17 and a lower pressureregion 10 in fluid communication with each other through channel 19 ofand extension 28 of inlet 18. Extension 28 with conical opening ofchannel 19 into region 17 provides a larger area to cover sample 29 onsubstrate 14. Substrate 14 is one embodiment is a glass microscope slidewhich is transparent to the laser beam 35 from the laser 40. Placing thesubstrate 14 against the conical extension 28 produces an imperfect sealbetween 28 and 14 exposing the sample 29 to a pressure lower than inregion 17. By using a matrix such as 2,5-DHAP which needs to be warmedto act as a MAIV matrix, a laser can be used to heat the matrix in thelocalized area by transmitting the laser beam through a focusing lens 34so that it focuses on the sample 29. The area of the sample heated byabsorption of the laser energy produces analyte ions. By rastering thelaser beam across the sample while acquiring mass spectra, images of thelocation of compounds in the sample 29 can be obtained using imagingsoftware. The sample in some embodiments is a section of biologicaltissue.

FIG. 9 illustrates the MAIV mass spectrum of bovine serum albumin (BSA)(Molecular weight [MW] ˜66000) using the vacuum MALDI source, hereafterthe vacuum source, of a Waters SYNAPT G2 ion mobility spectrometry (IMS)mass spectrometry (MS) instrument without the laser and using 3-NBN asthe matrix.

FIG. 10 (top) illustrates MAIV of lysozyme (MW 14300) and (bottom) itscomplex with a small peptide (PNAP) analyzed from buffered solutionusing 3-NBN as matrix on the vacuum source.

FIG. 11 illustrates MAIV of the fragile non volatile compounds: (top)GT1b gangliosides (MW 2128 and 2156) and (bottom) a phosphorylatedpeptide (MW 1332) using the vacuum source and negative mode detection.

FIG. 12 illustrates MAIV of the membrane protein bacteriorhodopsin (MW27000) 3-NBN as matrix using the vacuum source. Multiply charged ionsare observed (top). It should be understood that deconvolution programscan be used as is exemplified in the bottom mass spectrum.

FIG. 13 illustrates MAIV of a variety of analytes using the vacuumsource. The left panel shows the positive and the right panel thenegative mode measurements using 3-NBN as the matrix.

FIG. 14 illustrates MAIV using the vacuum source with 3-NBN as thematrix and 50 fmol of ubiquitin. The inset illustrates the isotopicdistribution of charge state +10 of ubiquitin.

FIG. 15 illustrates analyses of 5 femtomoles of clozapine. The leftpanel is the MAIV results using 3-NBN as matrix on the vacuum source andthe right panel using ESI

FIG. 16 illustrates MAIV using 3-NBN as matrix on the vacuum sourceusing a capillary as the substrate into which the sample was dried toprolong ionization. Ionization is extended from about 1 minute when the3-NBN matrix is applied to a traditional MALDI target plate to about 30minutes in the capillary.

FIG. 17 illustrates another method for extending the time of ionizationusing 3-NBN with vacuum source MAIV. The left panel illustrates thetotal ion chronograms and the right panel the mass spectra. The top rowwas obtained when the substrate was loaded into the vacuum source atroom temperature and the bottom row illustrates when the substrate(metal) was cooled to −80° C. and immediately introduced to the vacuumsource. Ionization is extended from about 1 minute to 11 minutes.

FIG. 18 illustrates MAIV using the vacuum source and the substrate at−80° C. to obtain the MS/MS spectrum of clozapine (MW 328) at 900attomoles deposited on the metal plate.

FIG. 19 illustrates MAIV mass spectra using the ESI source and thematrix 3-NBN and paper chromatography to separate compounds in a markerpen: (1) MS and (2) MS/MS of (A) red and (B) blue ink marked on filterpaper with 3-NBN; (I) analyzed as individual spots, (II) photograph ofthe filter paper after separation of the two inks, and (III) analysisafter separation: (1) MS and (2) MS/MS.

FIG. 20 illustrates MAIV from (top) a thin layer chromatography (TLC)plate and (bottom) a metal foil using the vacuum source and 3-NBN asmatrix.

FIG. 21 illustrates MAIV of a peptide using 3-NBN as matrix and thevacuum source. (top) low voltages applied and (bottom) extractionvoltages applied in the vacuum source.

FIG. 22 illustrates MAIV applying the matrix 3-NBN to a small area of a20 dollar bill using the vacuum source. Only the one eye of thePresident was marked with the matrix and analyzed by IMS-MS and MS/MSidentifying a polymer (PPG) and the presence of cocaine. Thisexemplifies the sensitivity and spatial analyses of the MAIV method.

FIG. 23 illustrates MAIV using the vacuum source and 3-NBN as matrix.Ions are instantaneously formed and separated as well as detected(IMS-MS). Charge states fall into families and detailed information canbe extracted (drift times, m/z). For example, the mass spectra of all 6components in the mixture of small drugs to proteins are obtained withlittle interference from each other.

FIG. 24 illustrates the MAIV mass spectra of a delipified tissue sectionusing the 3-NBN matrix and the vacuum source. The left panel shows apicture of deposition of the matrix using a micropipette, the IMS-MS 2-Dplot with inset mass spectra and indication of the respective chargestate families and the right panel shows tissue (top) before and(bottom) after MAIV-IMS-MS analyses providing spatial analyses.

FIG. 25 illustrates MAIV of drug dosed urine characterizing Cetirizine(MW 388) using the vacuum source and 3-NBN as the MAIV matrix (metalplate substrate). Top: mass spectrum and bottom MS/MS (CID) fragmentspectrum. It should be understood that in-source fragmentation can beused for certain applications.

FIG. 26 illustrates MAIV of 1-D gel (gel slices analyzed after gelelectrophoresis) using the vacuum source. After gel separation, the gelpiece is adhered to the plate through a mesh and the matrix pipettedinto the gel for extraction of the protein out of the gel and directprotein analyses by IMS-MS.

FIG. 27 illustrates MAIV using the vacuum source of (top) a syntheticpolymer (PEGDME 2000) using LiCl and (bottom) ubiquitin (MW 8561) and 1M NaCl as salt additives to the sample solution using 3-NBN as thematrix. The insets show the isotopic distribution of charge states.Synthetic polymers frequently depend on salt addition for forming ionsin MS and are useful with MAIV. The protein is robustly analyzed by MAIVin the presence of high salt content and desirably as protonated ions.

FIG. 28 illustrates the MAIV analyses of (top) a wet sample spot and(bottom) completely dried sample spot using 3-NBN as the matrix prior tointroduction to the vacuum source.

FIG. 29 illustrates eight MAIV matrices using the vacuum source. Thematrix structure used is indicated in each mass spectrum. Seven sampleswere prepared solvent-based using the droplet and three solvent-free.The total ion chronogram's (TIC's) (right) provide an indication of theduration of the ionization process.

FIG. 30 illustrates MAIV of lysozyme (MW 14300) using a binary matrixmixture of 3-NBN:CHCA 1.6:1 and (top) the vacuum source or (bottom) theAP source.

FIG. 31 illustrates MAIV of whole blood extracted from a band aid usingthe modified ESI skimmer cone (from here on referred to as the APsource). A picture of the design using a glass plate (left), the IMS-MS2-D plot of drift time versus m/z (middle) exemplifying the output ofcombining instantaneous ionization with instantaneous separation anddetection, and the cleanly separated beta chain of hemoglobin obtainedthrough the IMS separation (right). A unique pictorial ‘snapshot’ isobtained of the biological matrix blood.

FIG. 32 illustrates using the source displayed in FIG. 31 to analyze byMAIV-IMS-MS a mixture of five drugs. Top: Mass spectrum (left) andIMS-MS 2-D plot (right). The extracted drift times and m/z values aresufficiently distinctive to identify compounds using this so callednested dataset. Middle: the entire set of ions is fragmented (CID)simultaneously without precursor selection and MS/MS data extracted foreach compound and characterization achieved. MS/MS total mass spectrum(left) and IMS-MS 2-D plot (right). Bottom: the extracted MS/MS datasetis displayed for cocaine: mass spectrum (left) extracted from experimentwithout precursor selection and (right) traditional CID with precursorselection including the respective 2-D plot (far right) with an insetfor fragmentation path.

FIG. 33 illustrates MAIV of 40 fmol clozapine and 20 pmol bovine insulinusing 3-NBN. Incorporating the IMS dimension increases the dynamic rangeof the experiment.

FIG. 34 illustrates MAIV of nicotine and nornicotine using 3-NBN.

Incorporating the IMS dimension separates isobaric molecules ofnornicotine and 3-NBN.

FIG. 35 illustrates MAIV of lysozyme using 3-NBN from a bufferedsolution showing the mass spectra (top) in which the solution of analytewas added to filter paper and while wet the matrix in a separatesolution was added, and (bottom) the matrix solution was added to filterpaper and dried. To this precoated matrix layer the analyte was added.

FIG. 36 illustrates MAIV using 3-NBN of four different samples in lessthan 1 minute using the AP source sliding the paper strip, onto whichthe sample was applied, across the inlet opening to createsub-atmospheric pressure at the sample. A picture of the design is shown(top left), the TIC is shown (top right), and the mass spectra is shown(bottom).

FIG. 37 illustrates MAIV using (top) a pipet tip as a sample substrateas demonstrated in FIG. 7. A picture of the method (top) and the massspectrum (bottom) of carbonic anhydrase (MW ˜29,000) using the solventsystem 1:1 acetonitrile:water with 3-NBN as the matrix. Alternatively(bottom) a glass plate substrate (top) is used to analyze a fatty acid(MW 280) in the negative mode using the solvent system methanol with the3-NBN.

FIG. 38 illustrates MAIV with 3-NBN as the matrix using a 8-channelmultiple pipet tips dispenser.

FIG. 39 illustrates MAIV and the 3-NBN matrix of LSD (MW 323) using theAP source with a nozzle extension analyzing the sample from the bottomof one well of the well plate similar to FIG. 6.

FIG. 40 illustrates MAIV and the 3-NBN matrix of nicotine (MW 162) usingthe AP source analyzing the sample from well of the 384-well plate(pictures top left). 1, 3 and 5 μL of 5000 ng mL⁻¹ nicotine weredeposited in separate well. This increase is reflected in the TIC. Thenearly background free mass spectra are shown to the bottom right.

FIG. 41 illustrates MAIV analyses of Clarithromycin (MW 747) directlyfrom a tablet using the 3-NBN matrix and the AP source.

FIG. 42 illustrates MAIV using the AP source and 3-NBN as the matrix.Top: Picture and TIC provide view of the set up and the TIC's (right)provide an indication of the duration of the ionization process. Astream of gas close to the sample aids in ionization as shown in themiddle Figure; bottom is the result of no airflow provided.

FIG. 43 illustrates MAIV-ETD using the AP source. Top: Ubiquitin chargestate +11 on Orbitrap using the glass plate as the substrate with avacuum leak; Middle: charge state +4 of myelin basic protein (MBP) onLTQ Velos using a paper (see picture to the right) as the substrate;Bottom: Galanin charge state +5 on SYNAPT G2 using foil as thesubstrate. Fragment ions are sorted in the IMS dimension according tocharge states (not shown) providing speed in acquisition and analyses.

FIG. 44 illustrates MAIV using 3-NBN of five different samples in lessthan 3 seconds using a gently adjusted laser to heat the sample close tothe skimmer cone similar to FIG. 8. It should be understood that thelaser can be aligned in transmission or reflection geometry relative tothe sample to be heated.

FIG. 45 illustrates nine MAIV matrices using the AP source. Thestructure and temperature used is indicated in each mass spectrum. TheTIC's (right) provide an indication of the duration of the ionizationprocess. A glass substrate was used as is exemplified in FIG. 6.

FIG. 46 illustrates a MAIV matrix/temperature study (in the range of 30°C. to 450° C.) using the 3-NBN, 2,5-DHAP, 2-NPG,methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, and6-nitro-o-anisonitrile matrixes and the AP source of the SYNAPT G2(skimmer cone source, maximum temperature that can be applied is 150°C.) and the LTQ Velos (inlet tube, maximum temperature that can beapplied is 500° C.).

FIG. 47 illustrates MAIV-IMS-MS: (right panel) 2-D plot display of drifttime vs. m/z and (left panel) mass spectra of beta-amyloid (1-42) (MW4511 Da) using MAIV with 3-NBN matrix in (I) positive mode and (II)negative mode on the intermediate pressure vacuum source of SYNAPT G2,and (Ill) ESI comparison in negative mode. Insets show extracted drifttimes useful for cross-section analyses directly from its biological orsynthetic (not shown) environments. The advantage of using MAIV matricesin addition of the simplicity, sensitivity, robustness, and norequirement for applied energy or force is that the matrices sublime orevaporate when placed under vacuum conditions commonly used with massspectrometers and vacuum operated ion mobility spectrometers. Thus, thematrix will not accumulate on the lens elements and contaminate theinstrument as is the case with MALDI, LSII, and MAII.

Although the systems and methods have been described in terms ofexemplary embodiments, they are limited thereto. Rather, the appendedclaims should be construed broadly, to include other variants andembodiments of the systems and methods, which may be made by thoseskilled in the art without departing from the scope and range ofequivalents of the systems and methods.

1.-39. (canceled)
 40. A method of ionizing an analyte for analysis byion mobility spectrometry or mass spectrometry, comprising: providing asample comprising an analyte and a matrix, wherein the matrix is3-nitrobenzonitrile, 2-nitrobenzonitrile, 5-methyl-2-nitrobenzonitrile,coumarin, methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate,2-bromo-2-nitropropane-1,3-diol, 3-nitrobenzaldehyde,6-nitro-o-anisonitrile, phthalic anhydride, or mixtures thereof;producing gas phase positive or negative ions of the analyte withoutirradiating the sample with a laser and without exposing the sample tohigh velocity particles to produce the gas phase positive or negativeions of the analyte.
 41. The method of claim 40, further comprising thestep of cooling or heating the sample to a temperature between −80° C.and 150° C.
 42. The method of claim 41, wherein the temperature isbetween 25° C. and 80° C.
 43. The method of claim 40, further comprisingthe step of exposing the sample to sub-atmospheric pressure.
 44. Themethod of claim 43, wherein said sub-atmospheric pressure is between 750mm Hg and 1×10⁻⁷ Hg.
 45. The method of claim 40, wherein said gas phasepositive or negative ions of the analyte are singly or multiply charged.46. The method of claim 40, wherein the step of producing gas-phasepositive or negative ions of the analyte is a spontaneous process. 47.The method of claim 40, wherein said matrix sublimes or evaporates whenexposed to sub-atmospheric pressure.
 48. The method of claim 40, whereinsaid matrix sublimes or evaporates when exposed to sub-atmosphericpressure at a temperature of less than 70° C.
 49. The method of claim40, wherein said analyte is a biofilm, biological tissue, biologicalmaterial, edible goods, polymers, paintings, synthetic compounds,archeological artifacts, artificial bone, skin, urine or blood.
 50. Themethod of claim 40, wherein said analyte is selected from proteins,protein complexes, receptors, ligands, polymers, catalysts, lipids,drugs, metabolites, pesticides, peptides, chemically orpost-translationally modified peptides or proteins, DNA, RNA,carbohydrates, glycans, antibodies, biomarkers or compounds produced bysynthesis.
 51. The method of claim 40, further comprising the step ofplacing said sample on a substrate.
 52. The method of claim 51, whereinsaid substrate is comprised of tissue, metal, paper cloth, ribbon,glass, plastic, polymers, sodium dodecyl sulfate gel, paperchromatography plate, silica plate or woven fiber.
 53. The method ofclaim 51, wherein the substrate is the sample.
 54. The method of claim40, wherein said sample further comprises a solvent.
 55. The method ofclaim 54, wherein said solvent is water, methanol, ethanol, isopropanol,acetonitrile, tetrahydrofuran, chloroform, dimethylformamide, dimethylsulfoxide, acetone, or mixtures thereof.
 56. The method of claim 40,wherein said sample is prepared by mixing or grinding the analyte andmatrix together,
 57. The method of claim 40, wherein said sample is asolid.
 58. The method of claim 40, wherein said sample is in a frozenstate.
 59. The method of claim 40, wherein said sample further comprisesan ammonium salt, metal salt, acid, base or buffer.